A number of proteins and peptides that are normally synthesized by mammalian cells have proven to have medical, agricultural and industrial utility. These proteins and peptides may be of different molecular size and have a number of different functions, for example, they may be enzymes, structural proteins, growth factors and hormones. In essence both proteins and peptides are composed of linear sequences of amino acids which form secondary and tertiary structures that are necessary to convey the biological activity. Human parathyroid hormone has a relatively small molecular weight, which has made it possible to synthesize the peptide chemically by the sequential addition of amino acids. Thus, parathyroid hormone is commercially available, but in very small quantities at high cost. As a result, there is no human parathyroid hormone available at a reasonable price to supply the many potential medical, agricultural and industrial applications.
During the past ten years, microbiological techniques employing recombinant DNA have made it possible to use microorganisms for the production of species-different peptides. The microorganism is capable of rapid and abundant growth and can be made to synthesize the foreign product in the same manner as bacterial peptides. The utility and potential of this molecular biological approach has already been proven by microbiological production of a number of human proteins that are now available for medical and other uses.
Parathyroid hormone (PTH) is one of the most important regulators of calcium metabolism in mammals and is also related to several diseases in humans, animals, e.g. milk fever, acute hypocalsemia and otherwise pathologically altered blood calcium levels. This hormone therefore will be important as a part of diagnostic kits and will also have potential as a therapeutic in human and veterinary medicine.
The first synthesis of DNA for human preproparathyroid hormone was described by Hendy, G. N., Kronenberg, H. M., Potts, Jr. J. T. and Rich, A. 78 Proc. Natl. Acad. Sci. 7365-7369 (1981). DNA complementary in sequence to PTH mRNA was synthesized and made double stranded (Hendy et al. supra). This cDNA was cloned in pBR 322 DNA and E. coli X1776 was transfected. Of the colonies with correct antibiotic resistance, 23 out of 200 clones were identified as containing specific human PTH cDNA inserts. However, none of the 23 human PTH clones contained the full length insert (Hendy et al., supra). Later Breyel, E., Morelle, G., Auf'mkolk, B., Frank, R., Blocker, H. and Mayer, H., Third European Congress on Biotechnology, 10-14 Sep. 1988, Vol. 3, 363-369 described the presence of the human PTH gel in a fetal liver genomic DNA library constructed in the phage Charon 4A. A restriction enzyme fragment of the PTH gene was recloned and transfected into E. coli.
However, the work of Breyel, supra, demonstrate that E. coli degrades human PTH. Thus, a microorganism which shows a stable production of intact human parathyroid hormone has so far not been described. Further, parethyroid hormone has never before been isolated from yeast.